The paralogous transcriptional activators, MarA, SoxS and Rob, activate a common set of promoters, the marA/soxS/rob regulon of Escherichia coli, by binding a cognate site (marbox) upstream of each promoter. The extent of activation varies from one promoter to another and is only poorly correlated with the in vitro affinity of the activator for the specific marbox. Here, we examine the dependence of promoter activation on the level of activator in vivo by manipulating the steady-state concentrations of MarA and SoxS in Lon protease mutants and measuring promoter activation using lacZ transcriptional fusions. We found that: (i) the MarA concentrations needed for half-maximal stimulation varied by at least 19-fold among the 10 promoters tested; (ii) most marboxes were not saturated when there were 24,000 molecules of MarA per cell; (iii) the correlation between MarA concentration needed for half-maximal promoter activity in vivo with marbox binding affinity in vitro was poor and (iv) the two activators differed in their promoter activation profiles. The marRAB and sodA promoters could both be saturated by MarA and SoxS in vivo. However, saturation by MarA resulted in greater marRAB and lesser sodA transcription than did saturation by SoxS implying that the two activators interact with RNAP in different ways at the different promoters. Thus, the concentration and nature of activator determines which regulon promoters are activated and the extent of their activation.
gene regulation; AraC protein family; stress response
Transcriptional activation of the promoters of the mar/soxRS regulons by the sequence-related but independently inducible MarA and SoxS proteins renders Escherichia coli resistant to a broad spectrum of antibiotics and superoxide generators. Here, the effects of MarA and SoxS on transcription of the marRAB promoter itself were assayed in vitro by using a minimal transcription system and in vivo by assaying beta-galactosidase synthesized from marR::lacZ fusions. Purified MarA and MalE-SoxS proteins stimulated mar transcription about 6- and 15-fold, respectively, when the RNA polymerase/DNA ratio was 1. Purified MarA bound as a monomer to a 16-bp "marbox" located 69 to 54 nucleotides upstream of a putative RNA initiation site. Deletion of the marbox reduced MarA-mar binding 100-fold, abolished the stimulatory effects of MarA and SoxS on transcription in vitro, and reduced marR::lacZ synthesis about 4-fold in vivo. Deletion of upstream DNA adjoining the marbox reduced MarA binding efficiency 30-fold and transcriptional activation 2- to 3-fold, providing evidence for an accessory marbox. Although MarA and the mar operon repressor, MarR, bound to independent sites, they competed for promoter DNA in band shift experiments. Assays of marR::lacZ transcriptional fusions in marRAB deletion or soxRS deletion strains showed that the superoxide generator paraquat stimulates mar transcription via soxRS and that salicylate stimulates mar transcription both by antagonizing MarR and by a MarR-independent mechanism. Thus, transcription of the marRAB operon is autorepressed by MarR and autoactivated by MarA at a site that also can be activated by SoxS.
The MarA protein of Escherichia coli can both activate and repress the initiation of transcription, depending on the position and orientation of its degenerate 20-bp binding site (“marbox”) at the promoter. For all three known repressed genes, the marbox overlaps the promoter. It has been reported that MarA represses the rob promoter via an RNA polymerase (RNAP)-DNA-MarA ternary complex. Under similar conditions, we found a ternary complex for the repressed purA promoter also. These findings, together with the backwards orientation of repressed marboxes, suggested a unique interaction of MarA with RNAP in repression. However, no repression-specific residues of MarA could be found among 38 single-alanine replacement mutations previously shown to retain activation function or among mutants from random mutagenesis. Mutations Thr12Ala, Arg36Ala, Thr95Ile, and Pro106Ala were more damaging for activation than for repression, some up to 10-fold, so these residues may play a specific role in activation. We found that nonspecific binding of RNAP to promoterless regions of DNA was presumably responsible for the ternary complexes seen previously. When RNAP binding was promoter specific, MarA reduced RNAP access to the rob promoter; there was little or no ternary complex. These findings strongly implicate steric hindrance as the mechanism of repression of rob by MarA.
The AraC family transcription factor MarA activates ∼40 genes (the marA/soxS/rob regulon) of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.
When environmental conditions change, cell survival can depend on sudden production of proteins that are normally in low demand. Protein production is controlled by transcription factors which bind to DNA near genes and either increase or decrease RNA production. Many puzzles remain concerning the ways transcription factors do this. Recently we collected data relating the intracellular level of a single transcription factor, MarA, to the increase in expression of several genes related to antibiotic and superoxide resistance in Escherichia coli. These data indicated that target genes are turned on in a well-defined order with respect to the level of MarA, enabling cells to mount a response that is commensurate to the level of threat detected in the environment. Here we develop a computational model to yield insight into how MarA turns on its target genes. The modeling suggests that MarA can increase the frequency with which a transcript is made while decreasing the overall presence of the transcription machinery at the start of a gene. This mechanism is opposite to the textbook model of transcriptional activation; nevertheless it enables cells to respond quickly to environmental challenges and is likely of general importance for gene regulation in E. coli and beyond.
The Rob protein, isolated on the basis of its ability to bind to the right arm of the Escherichia coli origin of chromosomal replication, is about 50% identical in amino acid sequence to SoxS and MarA, the direct regulators of the superoxide (soxRS) and multiple antibiotic resistance (mar) regulons, respectively. Having previously demonstrated that SoxS (as a MalE-SoxS fusion protein) and MarA are essentially identical in their abilities to activate in vitro transcription of genes of the sox-mar regulons, we investigated the properties of Rob as a transcriptional activator. We found that Rob (i) activates the transcription of zwf,fpr,fumC, micF, nfo, and sodA, (ii) requires a 21-bp soxbox-marbox-robbox sequence to activate zwf transcription, (iii) protects the soxbox/marbox/robbox from attack by DNase 1, (iv) is ambidextrous, i.e., requires the C-terminal domain of the alpha subunit of RNA polymerase for activation of zwf but not fumC or micF, (v) bends zwf and fumC DNA, and (vi) binds zwf and fumC DNA as a monomer. Since these transcription activation properties of Rob are virtually identical to those of MalE-SoxS and MarA, it appears as if the E. coli genome encodes three genes with the same functional capacity. However, in contrast to SoxS and MarA, whose syntheses are induced by specific environmental stimuli and elicit a clear defense response, Rob is expressed constitutively and its normal function is unknown.
Elevated levels of fluoroquinolone resistance are frequently found among Escherichia coli clinical isolates. This study investigated the antibiotic resistance mechanisms of strain NorE5, derived in vitro by exposing an E. coli clinical isolate, PS5, to two selection steps with increasing concentrations of norfloxacin. In addition to the amino acid substitution in GyrA (S83L) present in PS5, NorE5 has an amino acid change in ParC (S80R). Furthermore, we now find by Western blotting that NorE5 has a multidrug resistance phenotype resulting from the overexpression of the antibiotic resistance efflux pump AcrAB-TolC. Microarray and gene fusion analyses revealed significantly increased expression in NorE5 of soxS, a transcriptional activator of acrAB and tolC. The high soxS activity is attributable to a frameshift mutation that truncates SoxR, rendering it a constitutive transcriptional activator of soxS. Furthermore, microarray and reverse transcription-PCR analyses showed that mdtG (yceE), encoding a putative efflux pump, is overexpressed in the resistant strain. SoxS, MarA, and Rob activated an mdtG::lacZ fusion, and SoxS was shown to bind to the mdtG promoter, showing that mdtG is a member of the marA-soxS-rob regulon. The mdtG marbox sequence is in the backward or class I orientation within the promoter, and its disruption resulted in a loss of inducibility by MarA, SoxS, and Rob. Thus, chromosomal mutations in parC and soxR are responsible for the increased antibiotic resistance of NorE5.
Multiple antibiotic resistance in Escherichia coli can be mediated by induction of the SoxS or MarA protein, triggered by oxygen radicals (in the soxRS regulon) or certain antibiotics (in the marRAB regulon), respectively. These small proteins (SoxS, 107 residues; MarA, 127 residues) are homologous to the C terminus of the XylS-AraC family of proteins and are more closely related to a approximately 100-residue segment in the N terminus of Rob protein, which binds the right arm of the replication origin, oriC. We investigated whether the SoxS-MarA homology in Rob might extend to the regulation of some of the same inducible genes. Overexpression of Rob indeed conferred multiple antibiotic resistance similar to that known for SoxS and MarA (against chloramphenicol, tetracycline, nalidixic acid, and puromycin), as well as resistance to the superoxide-generating compound phenazine methosulfate. The Rob-induced antibiotic resistance depended only partially on the micF antisense RNA that down-regulates the OmpF outer membrane porin to limit antibiotic uptake. Similar antibiotic resistance was conferred by expression of a Rob fragment containing only the N-terminal 123 residues that constitute the SoxS-MarA homology. Both intact Rob and the N-terminal fragment activated expression of stress genes (inaA, fumC, sodA) but with a pattern distinct from that found for SoxS and MarA. Purified Rob protein bound a DNA fragment containing the micF promoter (50% bound at approximately 10(-9) M Rob) as strongly as it did oriC, and it bound more weakly to DNA containing the sodA, nfo, or zwf promoter (50% bound at 10(-8) to 10(-7) M). Rob formed multiple DNA-protein complexes with these fragments, as seen previously for SoxS. These data point to a DNA-binding gene activator module used in different protein contexts.
Transcription of the multiple antibiotic resistance marRAB operon increases when one of the sequence-related activators, MarA, SoxS, or Rob, binds to the "marbox" centered at -61.5 relative to the transcriptional start site. Previous deletion analyses showed that an adjacent upstream "accessory region" was needed to augment the marbox-dependent activation. To analyze the roles of the marbox and accessory regions on mar transcription, thirteen promoters, each with a different 5-bp transversion of the -96 to -32 sequence, were synthesized, fused to lacZ, and assayed for beta-galactosidase production in single-copy lysogens with appropriate genotypes. The accessory region is shown here to be a binding site for Fis centered at -81 and to bind Fis, a small DNA-binding and -bending protein, with a Kd of approximately 5 nM. The binding of MarA to the marbox and that of Fis to its site were independent of each other. MarA, SoxS, and Rob each activated the mar promoter 1.5-to 2-fold when it had a wild-type marbox but Fis was absent. In the presence of MarA, SoxS, or Rob, Fis further enhanced the activity of the promoter twofold provided the promoter was also capable of binding Fis. However, in the absence of MarA, SoxS, or Rob or in the absence of a wild-type marbox, Fis nonspecifically lowered the activity of the mar promoter about 25% whether or not a wild-type Fis site was present. Thus, Fis acts as an accessory transcriptional activator at the mar promoter.
The Escherichia coli tolC encodes a major outer membrane protein with multiple functions in export (e. g., diverse xenobiotics, hemolysin) and as an attachment site for phage and colicins. tolC is regulated in part by MarA, SoxS and Rob, three paralogous transcriptional activators which bind a sequence called the marbox and which activate multiple antibiotic and superoxide resistance functions. Two previously identified tolC promoters, p1 and p2, are not regulated by MarA, SoxS or Rob but p2 is activated by EvgAS and PhoPQ which also regulate other functions. Using transcriptional fusions and primer extension assays, we show here that tolC has two additional strong overlapping promoters, p3 and p4, which are downstream of p1, p2 and the marbox and are activated by MarA, SoxS and Rob. p3 and p4 are configured so that a single marbox suffices to activate transcription from both promoters. At the p3 promoter, the marbox is separated by 20 bp from the −10 hexamer for RNA polymerase but at the p4 promoter, the same marbox is separated by 30 bp from the −10 hexamer. The multiple tolC promoters may allow the cell to respond to diverse environments by coordinating tolC transcription with other appropriate functions.
gene regulation; outer membrane protein; transcriptional start sites; efflux pumps; antibiotic resistance
The marRAB operon is a regulatory locus that controls multiple drug resistance in Escherichia coli. marA encodes a positive regulator of the antibiotic resistance response, acting by altering the expression of unlinked genes. marR encodes a repressor of marRAB transcription and controls the production of MarA in response to environmental signals. A molecular and genetic study of the homologous operon in Salmonella typhimurium was undertaken, and the role of marA in virulence in a murine model was assessed. Expression of E. coli marA (marAEC) present on a multicopy plasmid in S. typhimurium resulted in a multiple antibiotic resistance (Mar) phenotype, suggesting that a similar regulon exists in this organism. A genomic plasmid library containing S. typhimurium chromosomal sequences was introduced into an E. coli strain that was deleted for the mar locus and contained a single-copy marR'-'lacZ translational fusion. Plasmid clones that contained both S. typhimurium marR (marRSt) and marA (marASt) genes were identified as those that were capable of repressing expression of the fusion and which resulted in a Mar phenotype. The predicted amino acid sequences of MarRSt, MarASt, and MarBSt were 91, 86, and 42% identical, respectively, to the same genes from E. coli, while the operator/promoter region of the operon was 86% identical to the same 98-nucleotide-upstream region in E. coli. The marRAB transcriptional start sites for both organisms were determined by primer extension, and a marRABSt transcript of approximately 1.1 kb was identified by Northern blot analysis. Its accumulation was shown to be inducible by sodium salicylate. Open reading frames flanking the marRAB operon were also conserved. An S. typhimurium marA disruption strain was constructed by an allelic exchange method and compared to the wild-type strain for virulence in a murine BALB/c infection model. No effect on virulence was noted. The endogenous S. typhimurium plasmid that is associated with virulence played no role in marA-mediated multiple antibiotic resistance. Taken together, the data show that the S. typhimurium mar locus is structurally and functionally similar to marRABEc and that a lesion in marASt has no effect on S. typhimurium virulence for BALB/c mice.
Bacteria possess multiple mechanisms to survive exposure to various chemical stresses and antimicrobial compounds. In the enteric bacterium Escherichia coli, three homologous transcription factors—MarA, SoxS, and Rob—play a central role in coordinating this response. Three separate systems are known to regulate the expression and activities of MarA, SoxS, and Rob. However, a number of studies have shown that the three do not function in isolation but rather are coregulated through transcriptional cross talk. In this work, we systematically investigated the extent of transcriptional cross talk in the mar-sox-rob regulon. While the three transcription factors were found to have the potential to regulate each other's expression when ectopically expressed, the only significant interactions observed under physiological conditions were between mar and rob systems. MarA, SoxS, and Rob all activate the marRAB promoter, more so when they are induced by their respective inducers: salicylate, paraquat, and decanoate. None of the three proteins affects the soxS promoter, though unexpectedly, it was mildly repressed by decanoate by an unknown mechanism. SoxS is the only one of the three proteins to repress the rob promoter. Surprisingly, salicylate somewhat activates transcription of rob, while decanoate represses it a bit. Rob, in turn, activates not only its downstream promoters in response to salicylate but also the marRAB promoter. These results demonstrate that the mar and rob systems function together in response to salicylate.
OmpW is a minor porin whose biological function has not been clearly defined. Evidence obtained in our laboratory indicates that in Salmonella enterica serovar Typhimurium the expression of OmpW is activated by SoxS upon exposure to paraquat and it is required for resistance. SoxS belongs to the AraC family of transcriptional regulators, like MarA and Rob. Due to their high structural similarity, the genes under their control have been grouped in the mar/sox/rob regulon, which presents a DNA-binding consensus sequence denominated the marsox box. In this work, we evaluated the role of the transcription factors MarA, SoxS and Rob of S. enterica serovar Typhimurium in regulating ompW expression in response to menadione. We determined the transcript and protein levels of OmpW in different genetic backgrounds; in the wild-type and Δrob strains ompW was upregulated in response to menadione, while in the ΔmarA and ΔsoxS strains the induction was abolished. In a double marA soxS mutant, ompW transcript levels were lowered after exposure to menadione, and only complementation in trans with both genes restored the positive regulation. Using transcriptional fusions and electrophoretic mobility shift assays with mutant versions of the promoter region we demonstrated that two of the predicted sites were functional. Additionally, we demonstrated that MarA increases the affinity of SoxS for the ompW promoter region. In conclusion, our study shows that ompW is upregulated in response to menadione in a cooperative manner by MarA and SoxS through a direct interaction with the promoter region.
Expression of the marA or soxS genes is induced by exposure of Escherichia coli to salicylate or superoxides, respectively. This, in turn, enhances the expression of a common set of promoters (the mar/soxRS regulons), resulting in both multiple antibiotic and superoxide resistance. Since MarA protein is highly homologous to SoxS, and since a MalE-SoxS fusion protein has recently been shown to activate soxRS regulon transcription, the ability of MarA to activate transcription of these genes was tested. MarA was overexpressed as a histidine-tagged fusion protein, purified, cleaved with thrombin (leaving one N-terminal histidine residue), and renatured. Like MalE-SoxS, MarA (i) activated the transcription of zwf, fpr, fumC, micF, nfo, and sodA; (ii) required a 21-bp "soxbox" sequence to activate zwf transcription; and (iii) was "ambidextrous," i.e., required the C-terminal domain of the alpha subunit of RNA polymerase for activation of zwf but not fumC or micF. Thus, the mar and soxRS systems use activators with very similar specificities and mechanisms of action to respond to different environmental signals.
Paralogous transcriptional regulators MarA, Rob, and SoxS act individually and together to control expression of more than 80 Escherichia coli genes. Deletion of marA, rob, and soxS from an E. coli clinical isolate prevents persistence beyond 2 days postinfection in a mouse model of pyelonephritis. We used microarray analysis to identify 242 genes differentially expressed between the triple deletion mutant and its parent strain at 2 days postinfection in the kidney. One of these, znuC of the zinc transport system ZnuACB, displayed decreased expression in the triple mutant compared to that in the parental strain, and deletion of znuC from the parental strain reduced persistence. The marA rob soxS triple deletion mutant was less viable in vitro under limited-Zn and Zn-depleted conditions, while disruption of znuC caused a reduction in the growth rates for the parental and triple mutant strains to equally low levels under limited-Zn or Zn-depleted conditions. Complementation of the triple mutant with soxS, but not marA or rob, restored the parental growth rate in Zn-depleted medium, while deletion of only soxS from the parental strain led to low growth in Zn-depleted medium. Both results suggested that SoxS is a major regulator responsible for growth under Zn-depleted conditions. Gel shift experiments failed to show direct binding of SoxS to the znuCB promoter, thus suggesting indirect control of znuCB expression by SoxS. While SoxS expression in the triple mutant fully restored persistence, increased expression of znuACB via a plasmid in this mutant only partially restored wild-type levels of persistence in the kidney. This work implicates SoxS control of znuCB expression as a key factor in persistence of E. coli in murine pyelonephritis.
Efflux pumps function to rid bacteria of xenobiotics, including antibiotics, bile salts, and organic solvents. TolC, which forms an outer membrane channel, is an essential component of several efflux pumps in Escherichia coli. We asked whether TolC has a role during growth in the absence of xenobiotics. Because tolC transcription is activated by three paralogous activators, MarA, SoxS, and Rob, we examined the regulation of these activators in tolC mutants. Using transcriptional fusions, we detected significant upregulation of marRAB and soxS transcription and Rob protein activity in tolC mutants. Three mechanisms could be distinguished: (i) activation of marRAB transcription was independent of marRAB, soxR, and rob functions; (ii) activation of soxS transcription required SoxR, a sensor of oxidants; and (iii) Rob protein was activated posttranscriptionally. This mechanism is similar to the mechanisms of upregulation of marRAB, soxS, and Rob by treatment with certain phenolics, superoxides, and bile salts, respectively. The transcription of other marA/soxS/rob regulon promoters, including tolC itself, was also elevated in tolC mutants. We propose that TolC is involved in the efflux of certain cellular metabolites, not only xenobiotics. As these metabolites accumulate during growth, they trigger the upregulation of MarA, SoxS, and Rob, which in turn upregulate tolC and help rid the bacteria of these metabolites, thereby restoring homeostasis.
The multiple antibiotic resistance gene pqrA was cloned from the chromosomal DNA of a clinical isolate of Proteus vulgaris 881051 into Escherichia coli KY2563. The MICs of quinolones tetracycline, cephalosporin, and chloramphenicol for transformant strain DNS7020 were from 8 to 32 times higher than those for the parent strain, KY2563. The level of expression of outer membrane protein F (OmpF) by DNS7020 was lower than that of KY2563 but not as low as that of an OmpF-deficient control strain. The 1.4-kb fragment containing the pqrA gene had an open reading frame encoding a polypeptide of 122 amino acid residues with a molecular weight of about 14,000, which was consistent with the experimental value identified by the Maxicell method. The putative PqrA polypeptide showed significant amino acid sequence similarity to the E. coli proteins SoxS and MarA. These polypeptides are strongly conserved in predicted helix-turn-helix DNA binding domains. The MarA protein, which is responsible for multiple antibiotic resistance in E. coli, also decreases OmpF expression. Moreover, the SoxS protein, which is characterized as a superoxide response regulon of E. coli, has also been shown to increase resistance to many structurally unrelated antibiotics. The soxS gene increases superoxide dismutase levels in addition to decreasing OmpF expression. The expression level of superoxide dismutase with DNS7020 was about 1.5 times higher than that with KY2563. These findings suggest that the pqrA gene in P. vulgaris confers multidrug resistance in a way similar to that of the soxS and marA genes in E. coli.
The contribution of regulatory genes to fluoroquinolone resistance was studied with clinical Escherichia coli strains bearing mutations in gyrA and parC and with different levels of fluoroquinolone resistance. Expression of marA and soxS was evaluated by Northern blot analysis of isolates that demonstrated increased organic solvent tolerance, a phenotype that has been linked to overexpression of marA, soxS, and rob. Among 25 cyclohexane-tolerant strains detected by a screen for increased organic solvent tolerance (M. Oethinger, W. V. Kern, J. D. Goldman, and S. B. Levy, J. Antimicrob. Chemother. 41:111–114, 1998), we found 5 Mar mutants and 4 Sox mutants. A further Mar mutant was detected among 11 fluoroquinolone-resistant, cyclohexane-susceptible E. coli strains used as controls. Comparison of the marOR sequences of clinical Mar mutants with that of E. coli K-12 (GenBank accession no. M96235) revealed point mutations in marR in all mutants which correlated with loss of repressor function as detected in a marO::lacZ transcriptional assay. We found four other amino acid changes in MarR that did not lead to loss of function. Two of these changes, present in 20 of the 35 sequenced marOR fragments, identified a variant genotype of marOR. Isolates with the same gyrA and parC mutations showed increased fluoroquinolone resistance when the mutations were accompanied by overexpression of marA or soxS. These data support the hypothesis that high-level fluoroquinolone resistance involves mutations at several chromosomal loci, comprising structural and regulatory genes.
Escherichia coli K-12 strains are normally tolerant to n-hexane and susceptible to cyclohexane. Constitutive expression of marA of the multiple antibiotic resistance (mar) locus or of the soxS or robA gene product produced tolerance to cyclohexane. Inactivation of the mar locus or the robA locus, but not the soxRS locus, increased organic solvent susceptibility in the wild type and Mar mutants (to both n-hexane and cyclohexane). The organic solvent hypersusceptibility is a newly described phenotype for a robA-inactivated strain. Multicopy expression of mar, soxS, or robA induced cyclohexane tolerance in strains with a deleted or inactivated chromosomal mar, soxRS, or robA locus; thus, each transcriptional activator acts independently of the others. However, in a strain with 39 kb of chromosomal DNA, including the mar locus, deleted, only the multicopy complete mar locus, consisting of its two operons, produced cyclohexane tolerance. Deletion of acrAB from either wild-type E. coli K-12 or a Mar mutant resulted in loss of tolerance to both n-hexane and cyclohexane. Organic solvent tolerance mediated by mar, soxS, or robA was not restored in strains with acrAB deleted. These findings strongly suggest that active efflux specified by the acrAB locus is linked to intrinsic organic solvent tolerance and to tolerance mediated by the marA, soxS, or robA gene product in E. coli.
A genetic approach was undertaken to identify normal bacterial genes whose products function to limit the effective concentration of antibiotics. In this approach, a multicopy plasmid library containing cloned Escherichia coli chromosomal sequences was screened for transformants that showed increased resistance to a number of unrelated antibiotics. Three such plasmids were identified, and all contained sequences originating from the mar locus. DNA sequence analysis of the minimal complementation unit revealed that the resistance phenotype was associated with the presence of the marA gene on the plasmids. The putative marA gene product is predicted to contain a helix-turn-helix DNA binding domain that is very similar to analogous domains found in three other E. coli proteins. One such similarity was to the SoxS gene product, the elevated expression of which has previously been associated with the multiple antibiotic resistance (Mar) phenotype. Constitutive expression of marA conferred antibiotic resistance even in cells carrying a deletion of the chromosomal mar locus. We have also found that transformants bearing marA plasmids show a significant reduction in ompF translation but not transcription, similar to previously described mar mutants. However, this reduction in ompF expression plays only a minor role in the resistance mechanism, suggesting that functions encoded by genes unlinked to mar must be affected by marA. These results suggest that activation of marA is the ultimate event that occurs at the mar locus during the process that results in multiple antibiotic resistance.
A 7.8-kbp fragment of chromosomal DNA from a region controlling multiple antibiotic resistance (Mar) in Escherichia coli has been sequenced. Within the fragment is a potential divergent promoter region including marO, which contains two pairs of direct repeats, suggesting possible operator-regulatory sites. To the left of marO (region I) are one or two transcriptional units with three putative open reading frames (ORFs) encoding 64, 157, and 70 amino acids. To the right (region II) is a transcriptional unit containing three putative ORFs (ORF125/144, ORF129, and ORF72). Of six independent Mar mutants, four had mutations within the ORF encoding the first putative protein (ORF125/144) downstream of marO, including three different single-point mutations and an IS2 insertion. One of the other mutations occurred in marO (20-bp duplication), and the other occurred in a site in marO or ORF144 (a 1-bp change). All six mutations led to increased transcription of the region II transcript. High-copy-number plasmids containing marO and the adjacent ORF125/144 region from a wild-type source but not from a Mar mutant reduced the antibiotic resistance of a Mar mutant to levels comparable to those of wild-type cells. High-copy-number plasmids containing wild-type marO alone caused an increase in resistance to tetracycline, chloramphenicol, and norfloxacin in a wild-type strain. The nature of the Mar mutations and the results of the complementation studies suggest that ORF125/144 encodes a repressor (designated MarR) which acts at marO. The second ORF (ORF129), designated marA, would encode a protein, MarA, whose sequence shows strong similarity to those of a family of positive transcriptional regulators. A Tn5 insertion in marA inactivated the multiresistance phenotype of Mar mutants. The function of ORF72, designated marB, encoding the third putative protein in the operon, and that of other ORFs detected within the 7.8-kb fragment have not yet been determined.
Escherichia coli responds to oxidative stress by activating sets of coregulated genes that help the cell to maintain homeostasis. Identified previously by genetic and biochemical approaches, the soxRS system mediates the induction of 18 of these redox-inducible genes (including the soxS gene itself). An overlapping set of genes is activated by an assortment of structurally unrelated molecules with antibiotic activities; many genes in this response are controlled by the marRAB system. The activation of either the soxRS or the marRAB system results in enhanced resistance to both superoxide-generating agents and multiple antibiotics. In order to probe the extent of these regulatory networks, we have measured whole-genome transcriptional profiles of the E. coli response to the superoxide-generating agent paraquat (PQ), an inducer of the soxRS system, and to the weak acid salt sodium salicylate (NaSal), an inducer of the marRA system. A total of 112 genes was modulated in response to PQ, while 134 genes were modulated in response to NaSal. We have also obtained transcriptional profiles of the SoxS and MarA regulons in the absence of global stress, in order to establish the regulatory hierarchies within the global responses. Several previously unrelated genes were shown to be under SoxS or MarA control. The genetic responses to both environmental insults revealed several common themes, including the activation of genes coding for functions that replenish reducing potential; regulate iron transport and storage; and participate in sugar and amino acid transport, detoxification, protein modification, osmotic protection, and peptidoglycan synthesis. A large number of PQ- and NaSal-responsive genes have no known function, suggesting that many adaptive metabolic changes that ensue after stress remain uncharacterized.
MarR is the dedicated autorepressor of the marRAB operon found in seven genera of the Enterobacteraceae. The MarA transcriptional regulator directly activates numerous genes involved in multidrug resistance and other environmental responses. MarR is inactivated by certain phenolic ligands, such as salicylate, by an unknown mechanism. Our recent work has shown that several amino acid residues of Escherichia coli MarR affecting ligand binding are located between the dimerization and DNA-binding domains. To further characterize the ligand-binding region of MarR, we have now examined seven point mutants generated by random mutagenesis and eleven site-directed alanine replacement mutants for inactivation by three ligands: salicylate, 2,4-dinitrophenol, and plumbagin. Inactivation of MarR was quantitated in intact cells by loss of MarR-mediated repression of a chromosomal mar-lacZ transcriptional fusion. The results showed that most of the residues important for ligand effectiveness lay in the α1 and α2 helices of MarR, between the putative DNA-binding domain and the dimerization domain of MarR, reinforcing our earlier findings. Moreover, the three ligands had different, but overlapping, sets of residues impacting their effects on MarR.
ligand; repression; induction; mutagenesis; marRAB operon; error-prone PCR
The transcriptional activator Rob consists of an N-terminal domain (NTD) of 120 amino acids responsible for DNA binding and promoter activation and a C-terminal domain (CTD) of 169 amino acids of unknown function. Although several thousand molecules of Rob are normally present per Escherichia coli cell, they activate promoters of the rob regulon poorly. We report here that in cells treated with either 2,2"- or 4,4"-dipyridyl (the latter is not a metal chelator), Rob-mediated transcription of various rob regulon promoters was increased substantially. A small, growth-phase-dependent effect of dipyridyl on the rob promoter was observed. However, dipyridyl enhanced Rob's activity even when rob was regulated by a heterologous (lac) promoter showing that the action of dipyridyl is mainly posttranscriptional. Mutants lacking from 30 to 166 of the C-terminal amino acids of Rob had basal levels of activity similar to that of wild-type cells, but dipyridyl treatment did not enhance this activity. Thus, the CTD is not an inhibitor of Rob but is required for activation of Rob by dipyridyl. In contrast to its relatively low activity in vivo, Rob binding to cognate DNA and activation of transcription in vitro is similar to that of MarA, which has a homologous NTD but no CTD. In vitro nuclear magnetic resonance studies demonstrated that 2,2"-dipyridyl binds to Rob but not to the CTD-truncated Rob or to MarA, suggesting that the effect of dipyridyl on Rob is direct. Thus, it appears that Rob can be converted from a low activity state to a high-activity state by a CTD-mediated mechanism in vivo or by purification in vitro.
Multiple antibiotic resistance in Escherichia coli has typically been associated with mutations at the mar locus, located at 34 min on the E. coli chromosome. A new mutant, marC, isolated on the basis of a Mar phenotype but which maps to the soxRS (encoding the regulators of the superoxide stress response) locus located at 92 min, is described here. This mutant shares several features with a known constitutive allele of the soxRS gene, prompting the conclusion that it is a highly active allele of this gene. The marC mutation has thus been given the designation soxR201. This new mutant was used to examine the relationship between the mar and sox loci in promoting antibiotic resistance. The results of these studies indicate that full antibiotic resistance resulting from the soxR201 mutation is partially dependent on an intact mar locus and is associated with an increase in the steady-state level of mar-specific mRNA. In addition, paraquat treatment of wild-type cells is shown to increase the level of antibiotic resistance in a dose-dependent manner that requires an intact soxRS locus. Conversely, overexpression of MarA from a multicopy plasmid results in weak activation of a superoxide stress response target gene. These findings are consistent with a model in which the regulatory factors encoded by the marA and soxS genes control the expression of overlapping sets of target genes, with MarA preferentially acting on targets involved with antibiotic resistance and SoxS directed primarily towards components of the superoxide stress response. Furthermore, compounds frequently used to induce the superoxide stress response, including paraquat, menadione, and phenazine methosulfate, differ with respect to the amount of protection provided against them by the antibiotic resistance response.
Multiple factors control the expression of the outer membrane porins OmpF and OmpC in Escherichia coli. In this work, we investigated the role of the mar-sox-rob regulon in regulating outer membrane porin expression in response to salicylate. We provide both genetic and physiological evidence that MarA and Rob can independently activate micF transcription in response to salicylate, leading to reduced OmpF expression. MarA was also found to repress OmpF expression through a MicF-independent pathway. In the case of OmpC, we found that its transcription was moderately increased in response to salicylate. However, this increase was independent of MarA and Rob. Finally, we found that the reduction in OmpF expression in a tolC mutant is due primarily to Rob. Collectively, this work further clarifies the coordinated role of MarA and Rob in regulating the expression of the outer membrane porins.